Trisbenzimidazoles useful as topoisomerase I inhibitors

ABSTRACT

The present invention provides anti-neoplastic topoisomerase I inhibitors of the formula:  &lt;IMAGE&gt;  wherein Ar is (C6-C12)aryl or (6- to 12-membered) heteroaryl comprising 1-3 N, S or non-peroxide O, wherein N is unsubstituted or is substituted with (C1-C4)alkyl; X is H, CN, CHO, OH, acetyl, CF3, OCH3, NO2 or NH2; each Y is individually H, (C1-C4)alkyl or aralkyl; Y&#39; is H or (C1-C4)alkyl; n is 0 or 1; or a pharmaceutically acceptable salt therein.

BACKGROUND OF THE INVENTION

DNA topoisomerases are nuclear enzymes that control and modify thetopological states of DNA by catalyzing the concerted breaking andrejoining of DNA strands. See, for example, D'Arpa et al., Biochim.Biophys. Acta, 989, 163 (1989). Topoisomerase II enzymes alter thetopological state of DNA by means of a double strand break in the DNA.Mammalian topoisomerase II represents an effective pharmacologicaltarget for the development of cancer chemotherapeutics. (A. Y. Chen etal., Annu. Rev. Pharmacol. Toxicol., 34, 191 (1994)). Among the clinicalagents in use which are recognized as topoisomerase II inhibitors areetoposide (VP-16), teniposide (VM-26), mitoxantrone, m-AMSA, adriamycin(doxorubicin), ellipticine and daunomycin.

In comparison to topoisomerase II inhibitors, there are relatively fewknown topoisomerase I inhibitors. Camptothecin represents the mostextensively studied mammalian topoisomerase I inhibitor. See R. C. Galloet al., J. Natl. Cancer Inst., 46, 789 (1971) and B. C. Giovanella etal., Cancer Res., 51 3052 (1991). The broad spectrum of potentantineoplastic activity observed for camptothecin has prompted furtherefforts to identify other agents which can effectively poison mammaliantopoisomerase I.

It has recently been demonstrated that Hoechst 33342 (1),2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole,is an inhibitor of topoisomerase I. ##STR2##

This agent, which binds to the minor groove of DNA, traps the reversiblecleavable complex derived from DNA and topoisomerase I and produces alimited number of highly specific single-strand DNA breaks. For example,see A. Y. Chen et al., Cancer Res., 53, 1332 (1993) and A. Chen et al.,PNAS, 90, 8131 (1993). A limitation of Hoechst 33342 as an anticanceragent is the previously reported observation that it is not effectiveagainst tumor cell lines which overexpress MDR1. While KB 3-1 cells areknown to be quite sensitive to Hoechst 33342, with an IC₅₀ ofapproximately 9 nM, this compound is approximately 130-fold lesscytotoxic to KB V-1 cells, which are known to overexpress MDR1.Recently, several analogs of this bisbenzimidazole have beensynthesized, to further investigate the structure activity relationshipsassociated with their potency as topoisomerase I inhibitors and therelated cytotoxicity. For example, Q. Sun et al., Biorg. and Med. Chem.Lett., 4, 2871 (1994) disclosed the preparation of bis-benzamidazoles offormula (2): ##STR3## where n is 0, 1, 2, or 3. However, these compoundswere found to be about one order of magnitude less cytotoxic thanHoechst 33342. Therefore, a continuing need exists for new compoundsthat can induce DNA cleavage in the presence of mammalian topoisomeraseI.

SUMMARY OF THE INVENTION

The present invention provides a compound of general formula (I):##STR4## wherein Ar is aryl or a nitrogen-, sulfur- or oxygen-containingheteroaromatic group; X is H, CN, CHO, OH, acetyl, CF₃, O(C₁ -C₄)alkyl,NO₂ or NH₂ ; each Y is individually H, (C₁ -C₄)alkyl or aralkyl; Y' is Hor (C₁ -C₄) alkyl; n is 0 or 1; or a pharmaceutically acceptable saltthereof. Preferably, Ar is a (C₆ -C₁₂)aryl or a 5- to 12-memberedheteroaryl group comprising 1-3 N, S or non-peroxide O atoms in thering, wherein each N is unsubstituted or is substituted with (C₁-C₄)alkyl. As drawn, the Ar-group can occupy any position of the benzomoiety, i.e., positions 4-7, and X can occupy any available position onAr. When Ar is phenyl, preferably X is H or is a 4-substituent.

Preferably n is 1. When n is 0, X is preferably H, CN, or CHO. Y ispreferably H or CH₃. Y' is preferably H or CH₃.

Compounds of formula (I) are inhibitors of topoisomerase I, asdemonstrated by their ability to promote DNA cleavage in the presence oftopoisomerase I. Furthermore, compounds of formula (I) also arecytotoxic to mammalian tumor cells, including camptothecin-sensitive andcamptothecin-resistant tumor cells and tumor cell lines exhibitingmulti-drug resistance due to expression of the P-glycoprotein.

Therefore, the present invention also provides a method for theinhibition of mammalian tumor cell growth, comprising contacting asusceptible population of tumor cells with an effectivegrowth-inhibiting amount of a compound of formula (1), preferably incombination with a pharmaceutically acceptable carrier. The growth ofthe tumor cells can be inhibited in vitro, or vivo, by administering thecompound of formula (I) to a mammal in need of such treatment, such as ahuman cancer patient afflicted with a leukemia or solid tumor. Thecompounds of formula I can also be used to evaluate the activity oftopoisomerase I obtained from different sources, and are expected toexhibit at least some of the other bioactivities observed fortopoisomerase inhibitors, such as antibacterial, antifungal,antiprotozoal, antihelmintic and/or antiviral activity. For example,compound 14, shown on FIG. 1, exhibits antifungal activity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the synthesis of compounds 10-16.

FIG. 2 is a schematic depiction of the preparation of intermediates 4-8used to prepare compounds of the invention.

FIG. 3 is a schematic depiction of the preparation of intermediate 9.

DETAILED DESCRIPTION OF THE INVENTION

The aryl groups (Ar) useful in the present compounds comprise (C₆-C₁₈)aryl, preferably (C₆ -C₁₄) aryl, e.g., systems containing aromaticrings, which systems comprise a total of 6 to 12 carbon atoms. Thus, asused herein, the term "aryl" includes mono- or bis-(C₁-C₄)alkyl-substituted aryl, such as tolyl and xylyl; ar(C₁ -C₄)alkyl,such as benzyl or phenethyl; and alkaralkyl. Preferably aryl is phenyl,benzyl or naphthyl.

Heteroaromatic rings include aromatic rings containing up to 3 ringheteroatoms such as N, S or non-peroxide O, and up to 12 ring atoms.Representative aromatic rings include thiophene, benzothiophene,naphthothiophene, trianthrene, furan, benzofuran, isobenzofuran, pyran,chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole,pyridine, pyrazine, triazole, tetrazole, pyrazine, triazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, phenazine, isothiazole,phenothiazine, oxazole, isoxazole, furazan, phenoxazine and the like.Preferred heteroaromatic rings have a 5- or 6-membered heteroaromaticring which may or may not be fused to an aromatic ring such as a benzoring, e.g., the preferred 2-, 3- or 4-pyridyl substituents.

The term "alkyl" includes straight-chain or branched alkyl, as well ascycloalkyl and (cyloalkyl)alkyl, e.g., methyl, ethyl, i-propyl,cyclopropyl or cyclopropylmethyl.

Pharmaceutically acceptable salts include the acid addition salts ofbasic NH with organic or inorganic acids, e.g., hydrochloride,carbonate, sulfate, acetate, phosphate, tartarate, citrate, malate,maleate, propionate, and the like.

The preparation of representative substituted trisbenzimidazoles isoutlined in FIG. 1. With the exception of phenylenediamine which wascommercially available, the appropriately substituted phenylenediamineswere synthesized by catalytic hydrogenation of the respectiveo-nitroaniline derivatives. These phenylenediamines were then coupledwith 5-formyl-2-(benzimidazo-5'-yl)benzimidazole, 9, by heating innitrobenzene at 150° C. to provide the various trisbenzimidazoles,10-16, in yields ranging from 43-96%, employing the generalmethodologies of M. P. Singh et al., Chem. Res. Toxicol., 5, 597 (1992)and Y. Bathini et al., Synth Comm., 20, 955 (1990).

The requisite nitroanilines, as outlined in FIG. 1, with the exceptionof 3 which was commercially available, were synthesized from4-bromo-2-nitroaniline, 17. Compound 17 was prepared from o-nitroanilinein good yield, 94%, using 2,4,4,6-tetrabromo-2,5-cyclohexadienone as thebromination reagent. G. J. Fox et al., Org. Syn. 55, 20 (1973). Whileallyltributyltin and phenyltributyltin are commercially available, thepyridyltributyltin derivatives were prepared from tributyltin chlorideand 2-, 3-, and 4-bromopyridine, respectively. See D. Peters et al.,Heterocyclic Chem., 27 2165 (1990). These tributyltin derivatives werethen coupled with 4-bromo-2-nitroaniline using PdCl₂ (PPh₃)₂ as thecatalyst in DMF as outlined in FIG. 2 to provide compounds 4, 5, 6, 7,and 8, respectively, in accord with the methodology of M. Iwao et al.,Heterocycles, 36, 1483 (1993). This methodology can generally be appliedto prepare 3-, 4-, 5- or 6-aryl- and heteroaryl-substituted2-nitroanilines from the corresponding bromonitroanilines.

The preparation of 5-formyl-2-(benzimidazo-5'-yl)benzimidazole, 9, wasaccomplished as outlined in FIG. 3. Reduction of5-benzimidazolecarboxylic acid to 5-hydroxymethylbenzimidazole wasaccomplished using LiAlH₄. Oxidation of the resulting crude benzylicalcohol with tetrapropylammonium perruthenate (TPAP) andN-methylmorpholine N-oxide provided in two steps the desired5-formylbenzimidazole in 32% an overall yield. See, A. Cherif et al., J.Med. Chem., 35, 3208 (1992). Coupling of 5-formylbenzimidazole with4-cyano-1,2-phenylenediamine provided5-cyano-2-(benzimidazol-5'-yl)benzimidazole, 19, which when treated withNi--Al catalyst in the presence of aqueous formic acid gave5-formyl-2-(benzimidazol-5'-yl)benzimidazole, 9, in 65% yield. (J. R.Pipier et al., J. Med. Chem., 31, 2164 (1988)).

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host, suchas a human cancer patient, in a variety of forms adapted to the chosenroute of administration, i.e., orally or parenterally, by intravenously,intramuscularly or subcutaneous routes.

Thus, the present phosphoramidate compounds may be orally administered,for example, in combination with a pharmaceutically acceptable vehiclesuch as an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of the patient'sdiet. For oral therapeutic administration, the active compound may becombined with one or more excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 0.1% of active compound. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 2 to about 60% of the weight of a givenunit dosage form. The amount of active compound in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin or a flavoring agent such as peppermint,oil of wintergreen, or cherry flavoring may be added. When the unitdosage form is a capsule, it may contain, in addition to materials ofthe above type, a liquid carrier, such as a vegetable oil or apolyethylene glycol. Various other materials may be present as coatingsor to otherwise modify the physical form of the solid unit dosage form.For instance, tablets, pills, or capsules may be coated with gelatin,wax, shellac or sugar and the like. A syrup or elixir may contain theactive compound, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and flavoring such as cherry ororange flavor. Of course, any material used in preparing any unit dosageform should be pharmaceutically acceptable and substantially non-toxicin the amounts employed. In addition, the active compound may beincorporated into sustained-release preparations and devices.

The active compound may also be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion usecan include sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusable solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form must be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, and liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersion orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze-drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

Useful dosages of the compounds of 1 can be determined by comparingtheir in vitro activity, and in vivo activity in animal models, to thatof an equivalent dosage of camptothecin (see, for example, B. C.Giovanella et al., Cancer Res., 51, 3052 (1991)) or Hoechst 33342 (see,A. Y. Chen et al., Cancer Res., 53, 1332 (1993)). Methods for theextrapolation of effective anti-tumor dosages in mice, and otheranimals, to humans are known to the art; for example, see U.S. Pat. No.4,938,949.

The present analogs can be used to treat cancers known to be susceptibleto topoisomerase I inhibitors, including, but not limited to, Burkitt'stumor, chronic lymphocytic leukemia, multiple myeloma, squamous cell andlarge cell anaplastic carcinomas, adenocarcinoma of the lung, Ewing'ssarcoma, non-Hodgkins lymphoma, breast tumor, colon tumor, stomachtumor, oat-cell bronchogenic carcinoma, squamous cell carcinoma of thecervix, ovarian tumors, bladder tumors, testicular tumors, endometrialtumors, malignant melanoma and acute lymphocytic leukemia, and prostaticcarcinoma. The present compounds can be administered as single agents,or in combination with other antineoplastic drugs commonly employed totreat these cancers.

The invention will be further described by reference to the followingdetailed examples, wherein melting points were determined with aThomas-Hoover unimelt capillary melting point apparatus. Infraredspectral data (IR) were obtained on a Perkin-Elmer 1600 Fouriertransform spectrophotometer and are reported in cm⁻¹. Proton (¹ H NMR)and carbon (¹³ C NMR) nuclear magnetic resonance were recorded on aVarian Gemini-200 Fourier Transform spectrometer. NMR spectra (200 MHz ¹H and 50 MHz ¹³ C) were recorded in CDCl₃ (unless otherwise noted) withchemical shifts reported in δ units downfield from tetramethylsilane(TMS). Coupling constants are reported in hertz. Mass spectra wereobtained from Midwest Center for Mass Spectrometry within the Departmentof Chemistry at the University of Nebraska-Lincoln. Combustion analyseswere performed by Atlantic Microlabs, Inc., Norcross, Ga., and were within ±0.4%. THF was freshly distilled form sodium and benzophenone priorto use. Allyltributyltin and phenyltributyltin were purchased fromAldrich Chemical Company.

EXAMPLE 1 General Procedure for PdCl₂ (PPh₃)₂ -catalyzed CouplingReaction of 4-Bromo-2-nitroaniline (13) with Tin Compounds

(A) 4-Phenyl-2-nitroaniline (5). A solution of 4-bromo-2-nitroaniline 17(1.0 g, 4.67 mmol), tributylphenyl tin (2.2 g, 6.07 mmol),bis(triphenylphosphine)palladium (II) chloride (164 mg, 0.234 mmol), andtriphenylphosphine (613 mg, 2.34 mmol) in DMF (15 ml) was heated underN₂ at 120° C. overnight. After the solution was cooled to roomtemperature, the reaction mixture was directly chromatographed on silicagel eluting with 2-5% EtOAc/Hexane to give 752 mg (75%) of 5 as a yellowsolid: mp 169°-171 ° C.; IR (CHCl₃) 3517, 3398, 3022, 1635, 1525, 1250;¹ H NMR δ8.38 (1H, d, J=2.2), 7.66 (1H, dd, J=8.7, 2.2), 7.59-7.54 (2H,m), 7.49-7.34 (3H, m), 6.90 (1H, d, J =8.8), 6.13 (NH, brs); ¹³ C NMRδ144.2, 139.3, 135.0, 130.9, 129.5, 127.8, 126.8, 124.4, 119.8, 112.8;Anal. Calcd for C₁₂ H₁₀ N₂ O₂ : C, 67.28; H, 4.70; N, 13.08. Found: C,67.38, H, 4.76; N, 13.01.

(B) 4-Allyl-2-nitroaniline (4). Prepared from 4-bromo-2-nitroaniline 17(1.70 g, 7.84 mmol) and allyltributyltin (3.38 g, 10.2 mmol) as a yellowsolid in 96% yield as described above for 5: mp 29°-31° C.; IR (KBr)3490, 3374, 1638, 1518, 1341, 1253; ¹ H NMR δ7.90 (1H, d, J=2.0), 7.19(1H, dd, J=8.5, 2.0), 6.77 (1H, d, J=8,5), 6.05 (NH, brs), 6.00-5.80(1H, m), 5.11 (1H, dd, J=1.4, 1.4), 5.04 (1H, ddd, J=6.6, 3.0, 1.5),3.28 (1H, d, J=6.6); ¹³ C NMR δ143.81, 137.13, 129.34, 125.59, 119.49,116.95, 39.18; HRMS (EI) calcd for C₉ H₁₀ N₂ O₂ 178.0742, found178.0746.

(C) 4-(2'-Pyridyl)-2-nitroaniline (6). Prepared from4-bromo-2-nitroaniline 17 (597 mg, 2.75 mmol) and2-tributylstannylpyridine (1.01 g, 2.75 mmol) as a yellow solid in 52%yield as described above for 5: mp 146°-148° C.; IR (CHCl₃) 3516, 3397,3020, 1634, 1524, 1341, 1250; ¹ H NMR δ8.74 (1H, d, J =2.2), 8.63 (1H,dd, J=4.9, 1.5), 8.13 (1H, dd, J=8.8, 2.1), 7.78-7.66 (2H, m), 7.20 (1H,ddd, J=4.8, 4.7, 1.9), 6.92 (1H, d, J=8.8), 6.37 (NH, brs); ¹³ C NMRδ155.6, 150.1, 145.6, 137.4, 134.5, 129.1, 124.7, 122.4, 119.8, 119.7;Anal. Calcd for C₁₁ H₉ N₃ O₂ : 61.39; H, 4.21; N, 19.53. Found: C,61.29; H, 4.23; N, 19.43.

(D) 4-(3'-Pyridyl)-2-nitroaniline (7). Prepared from4-bromo-2-nitroaniline 17 (1.42 g, 6.53 mmol) and3-tributylstannylpyridine (3.60 g, 9.79 mmol) as a yellow solid in 32%yield as described above for 5: mp 177°-179° C.; IR (CHCl₃) 3515, 3399,3052, 2983, 1638, 1524, 1341, 1259; ¹ H NMR δ8.68 (1H, d, J=1.7), 8.42(1H, dd, J=4.8, 1.5), 8.22 (1H, d, J=2.2), 7.74 (1H, ddd, J=7.9, 2.4,1.6), 7.50 (1H, dd, J=8.7, 2.2), 7.23 (1H, ddd, J=8.0, 4.8, 0.8), 6.92(1H, d, J=8.8), 6.56 (NH, brs); ¹³ C NMR δ148.7, 147.8, 145.4, 135.0,134.4, 133.8, 126.5, 124.4, 124.0, 120.4; Anal. Calcd for C₁₁ H₉ N₃ O₂ :C, 61.39; H, 4.21; N, 19.53. Found: C, 61.28; H, 4.16; N, 19.40.

(E) 4-(4'-Pyridyl)-2-nitroaniline (8). Prepared from4-bromo-2-nitroaniline 17 (165 mg, 0.76 mmol) and4-tributylstannylpyridine (280 mg, 0.76 mmol) as a yellow solid in 25%yield as described above for 5: mp 230°-232° C.; IR (CHCl₃) 3518, 3398,3032, 1636, 1528, 1344; ¹ H NMR (CD₃ OD) δ8.55 (2H, d, J=6.3), 8.52 (1H,d, J=2.3), 7.84 (1H, dd, J=8.9, 2.3), 7.71 (2H, d, J=6.4), 7.13 (1H, d,J=8.9); ¹³ C NMR (CD₃ OD) δ149.4, 133.4, 124.0, 120.7, 120.0; HRMS (El)calcd for C₁₁ H₉ N₃ O₂ 215.0695, found 215.0698.

EXAMPLE 2 5-Formyl-2-(benzimidazol-5'-yl)benzimidazole (9)

A mixture of 5-cyano-2-(benzimidazol-5'-yl)benzimidazole 19 (148 mg,0.57 mmol), Ni--Al catalyst (500 mg), formic acid (7 ml) and water (3ml) was heated under refluxed under N₂ for 4 h. The hot reaction mixturewas immediately filtered through a plug of celite, and evaporated togive a yellow solid. The yellow solid was then dissolved in hot water (5ml), and the solution was neutralized to pH 9 by 2N NaOH. The solidprecipitated was collected by suction filtration and further purified byflash chromatography on silica gel (15% MeOH/EtOAc) to give 142 mg (95%)of 9 as a white solid: mp>275° C.; IR (KBr) 3106, 2835, 1685, 1618,1432, 1293; ¹ H NMR (CD₃ OD) δ10.01 (1H, s), 8.39 (1H, s), 8.35 (1H, s),8.13 (1H, s), 8.06 (1H, dd, J=8.6, 1.6), 7.83 (1H, dd, J=8.4, 1.4), 7.77(1H, d, J=8.5), 7.71 (1H, d, J=8.3); HRMS (FAB) calcd for C₁₅ H₁₁ N₄ O263.0933, found 263.0932.

EXAMPLE 3 General Procedures for Preparing 5-substitutedtrisbenzimidazoles

(A) 2- 2'-(Benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole (10). Amixture of 5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (121 mg, 0.46mmol) and phenylenediamine (60 mg, 0.55 mmol) in nitrobenzene (8 ml) washeated at 150° C. under N₂ overnight. The mixture was cooled to roomtemperature and chromatographed on silica gel (0-20% MeOH/EtOAc) toafford 155 mg (96%) of 10 as a solid: mp>275° C.; IR (KBr) 3400, 3157,1630, 1542, 1438, 1294; ¹ H NMR (DMSO-d₆ +3 drops of CF₃ COOH) δ9.71(1H, s), 8.75 (1H, s), 8.65 (1H, d, J=1.1), 8.48 (1H, dd, J=8.7, 1.5),8.21 (1H, dd, J=8.6, 1.6), 8.14 (1H, d, J=8.8), 8.08 (1H, d, J=8.7),7.90 (2H, dd, J=6.2, 3.1), 7.61 (2H, dd, J=6.1, 3.1); ¹³ C NMR (DMSO-d₆+3 drops of CF₃ COOH) δ154.4, 149.8. 133.2, 132.0, 131.7, 126.2, 125.5,125.4, 123.9, 123.6, 116.3, 115.9, 114.23, 114.17, 114.13; HRMS (FAB)calcd for C₂₁ H₁₅ N₆ 351.1358, found 351.1367.

(B) 5-Cyano-2- 2'-(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole(11). Hydrogenation of 3 (70 mg, 0.43 mmol) was accomplished at 40 psiH₂ at room temperature for 1 h using 10% Pd-C (30 mg) in EtOAc (10 ml).The reaction mixture was filtered and concentrated in vacuo to afford asolid. The solution of this solid and 9 (87 mg, 0.33 mmol) innitrobenzene (5 ml) was heated at 150° C. under N₂ overnight. Themixture was cooled to room temperature, and chromatographed directly onsilica gel (0-10% MeOH/EtOAc) to give 107 mg (86%) of 11 as a solid;mp>280° C.; IR (KBr) 3416, 3148, 2222, 1626, 1553, 1441, 1292; ¹ H NMR(DMSO-d₆ +3 drops of CF₃ COOH) δ8.50 (1H, s), 8.46 (1H, s), 8.40 (1H,s), 8.18-8.11 (3H, m), 7.81-7.75 (3H, m), 7.62 (1H, dd, J=8.3, 1.5);HRMS (FAB) calcd for C₂₂ H₁₃ N₇ 376.1310, found 376.1309.

(C) 5-Propyl-2- 2'-(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole(12). Prepared from 4-allyl-2-nitroaniline 4 (312 mg, 1.75 mmol) and5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (121 mg, 0.46 mmol) in79% yield as described above for 11: solid; mp>270° C.; IR (KBr) 3421,3068, 2957, 1434; ¹ H NMR (DMSO-d₆ +3 drops of CF₃ COOH) δ9.66 (1H, s),8.73 (1H, s), 8.59 (1H, s), 8.48 (1H, dd, J=8.7, 1.5), 8.13 (1H, dd,J=8.7, 1.4), 8.11 (1H, d, J=8.7), 8.02 (1H, d, J=8.5), 7.79 (1H, d,J=8.4), 7.66 (1H, s), 7.45 (1H, dd, J=8.5, 1.3), 2.80 (2H, t, J=7.0),1.70 (2H, m), 0.96 (3H, t, J=7.2); ¹³ C NMR (DMSO-d₆ +3 drops of CF₃COOH) δ153.84, 149.74, 141.64, 141.01, 139.37, 133.10, 132.26, 131.99,130.34, 127.08, 126.26, 125.14, 141.64, 141.01, 139.37, 133.10, 132.26,131.99, 130.34, 127.08, 126.26, 125.14, 122.91, 117.52, 116.32, 116.06,115.76, 113.78, 112.99, 37.45, 24.73, 13.74;

(D) 5-Phenyl-2- 2'(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole(13). Prepared from 4-phenyl-2-nitroaniline 5 (247 mg, 1.15 mmol) and5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (201 mg, 0.77 mmol) in89% yield as described for 11: solid; mp 262°-164° C. dec; IR (KBr)3402, 3104, 1627, 1552, 1442, 1290; ¹ H NMR (DMSO-d₆ +3 drops of CF₃COOH) δ9.66 (1H, s), 8.74 (1H, s), 8.65 (1H, s), 8.50 (1H, dd, J=8.8,1.1), 8.21 (1H, dd, J=8.7, 1.4), 8.12 (1H, d, J=8.8), 8.06 (1H, s), 8.05(1H, d, J=8.4), 7.97 (1H, d, J=8.7), 7.89 (1H, dd, J=8.7, 1.5), 7.80(2H, d, J=7.0), 7.61-7.47 (3H, m); HRMS (FAB) calcd for C₂₇ H₁₉ N₆427.1671, found 427.1666.

(E) 5-(2-Pyridyl)-2-2'-(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole (14). Preparedfrom 4-(2'-pyridyl)-2-nitroaniline, 6 (110 mg, 0.50 mmol), and5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (51 mg, 0.25 mmol) in 84%yield as described above for 11: solid; mp>275° C.; IR (KBr) 3411, 3157,1630, 1593, 1432; ¹ H NMR (CD₃ OD) δ8.59 (1H, d, J=4.8), 8.35 (1H, s),8.31-8.25 (2H, m), 8.10 (1H, s), 8.04-7.94 (2H, m), 7.85-7.77 (3H, m),7.72 (1H, d, J=8.6), 7.68 (1H, d, J=8.7), 7.64 (1H, d, J=8.7), 7.30 (1H,m); HRMS (FAB) calcd for C₂₆ H₁₈ N₇ 428.1624, found 428.1611.

(F) 5-(3-Pyridyl)-2-2'-(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole (15). Preparedfrom 4-(3'-pyridyl)-2-nitroaniline 7 (183 mg, 0.85 mmol) and5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 in 46% yield as describedabove for 11: solid; mp>275° C.; IR (KBr) 3400, 3070, 2836, 1438, 1289;¹ H NMR (CD₃ OD) δ8.83 (1H, d, J=1.6), 8.49 (1H, dd, J=4.9, 1.5), 8.38(1H, d, J=1.1), 8.31 (1H, d, J=1.1), 8.29 (1H, s), 8.11 (1H, ddd, J=8.0,2.3, 1.6), 8.05 (1H, dd, J=8.5, 1.6), 8.00 (1H, dd, J=8.5, 1.6), 7.81(1H, d, J=1.1), 7.77-7.68 (3H, m), 7.55-7.47 (2H, m); HRMS (FAB) calcdfor C₂₆ H₁₈ N₇ 428.1624, found 428.1612.

(G) 5-(4-Pyridyl)-2-2'-(benzimidazol-5"-yl)benzimidazol-5'-yl!benzimidazole (16). Preparedfrom 4-(4'-pyridyl)-2-nitroaniline 8 (35 mg, 0.16 mmol) and5-formyl-2-(benzimidazol-5'-yl)benzimidazole 9 (50 mg, 0.19 mmol) in 43%yield as described above for 11: solid; mp>280° C.; IR (KBr) 3411, 3118,1600, 1552, 1439, 1290; ¹ H NMR (CD₃ OD) δ8.51 (2H, d, J=6.2), 8.33 (1H,d, J=1.1), 8.27 (1H, s), 8.25 (1H, d, J=1.1), 8.01 (1H, dd, J=8.6, 1.7),7.96 (1H, dd, J=8.9, 2.0), 7.87 (1H, d, J=1.0), 7.74-7.56 (6H, m); HRMS(FAB) calcd for C₂₆ H₁₈ N₇ 428.1624, found 428.1625.

EXAMPLE 4 4-Bromo-2-nitroaniline (17)

A solution of 2-nitroaniline (5 g, 36.2 mmol) in CH₂ Cl₂ (100 ml) wascooled to -10° C., and treated by 90%2,4,4,6-tetrabromo-2,5-cyclohexadienone (19.8 g, 43.5 mmol) in 5portions. The mixture was stirred at -10° C.-0° C. for 1 hr. After beingwarmed to room temperature, the reaction mixture was washed by 2N NaOH(60 ml) and brine (50 ml), dried over Na₂ SO₄ and evaporated. Flashchromatography on silica gel (5% EtOAc/Hexane) gave 7.40 g (94%) of 17as a yellow solid: mp 109-110 (lit. mp 112°-113° C.); ¹ H NMR δ8.27 (1H,d, J=2.3), 7.43 (1H, dd, J=8.9, 2.4), 6.73 (1H, d, J=8.8), 6.09 (NH,brs).

EXAMPLE 5 5-Formylbenzimidazole (18)

A suspension of 5-benzimidazolecarboxylic acid (1.57 g, 9.7 mmol) in dryTHF (50 ml) was cooled to -78° C. under N₂, and treated with LiAlH₄ (736mg, 19.4 mmol). After the addition, the mixture was allowed to warmslowly to room temperature and then stirred at r.t. overnight. Themixture was quenched by MeOH and H₂ O cautiously, and passed through ashort silica gel column eluting with 10% MeOH/EtOAc. The eluate wasconcentrated to give 876 mg crude alcohol as a solid. The crude alcohol(876 mg) was dissolved in a mixture of DMF (3 ml), THF (10 ml) and CH₂Cl₂ (40 ml). 4-Methylmorpholine N-oxide (2.25 g, 19.2 mmol), 4Amolecular sieves (5 g), and TPAP (169 mg, 0.48 mmol) were subsequentlyadded to the crude alcohol solution. The mixture was stirred at roomtemperature overnight, and filtered through a pad of silica gel elutingwith 10% MeOH/EtOAc. The elute was concentrated and further purified byflash chromatography on silica gel eluting with 0-10% MeOH/EtOAc to give452 mg (32% 2 steps) of 17 as a white solid: mp 164°-166° C.; IR (KBr)3087, 2818, 1690, 1292; ¹ H NMR (CD₃ OD) δ9.95 (1H, s), 8.34 (1H, s),8.08 (1H, d, J=1.5). 7.74 (1H, dd, J=8.4, 1.5), 7.63 (1H, d, J=8.4); ¹³C NMR (CD₃ OD) δ194.2, 146.0, 143.0, 139.8, 133.6, 124.9, 120.7, 116.6;Anal. Calcd for C₈ H₆ N₂ O: C, 65.75; H, 4.14; N, 19.17. Found: C,65.60; H, 4.17; N, 19.08.

EXAMPLE 6 5-Cyano-2-(benzimidazol-5'-yl)benzimidazole (19)

A mixture of 5-formylbenzimidazole 18 (211 mg, 1.44 mmol) and4-cyano-1,2-phenylenediamine (230 mg, 1.73 mmol) in nitrobenzene (10 ml)was heated at 150° C. under N₂ overnight. The mixture was cooled to roomtemperature and directly chromatographed on silica gel eluting with0-15% MeOH/EtOAc to give 244 mg (65%) of 18 as a solid: mp >270° C.; IR(KBr) 3110, 2826, 2224, 1627, 1426, 1294; ¹ H NMR (CD₃ OD) δ8.41 (1H,s), 8.33 (1H, s), 8.07 (1H, dd, J=8.6, 1.5), 7.98 (1H, s), 7.78 (1H, d,J=8.4), 7.73 (1H, d, J=8.4), 7.56 (1H, dd, J=8.4, 1.5); 13C NMR (DMSO-d₆+3 drops of CF₃ COOH) δ153.4, 140.4, 138.3, 132.9, 131.6, 127.0, 125.8,125.3, 120.8, 119.8, 116.0, 115.8, 113.9, 105.5; HRMS (FAB) calcd forC₁₅ H₁₀ N₅ 260.0936, found 260.0935.

EXAMPLE 7 Bioassays A. Topoisomerase I-Mediated DNA Cleavage Assays

DNA topoisomerase I was purified from calf thymus gland as reportedpreviously by B. D. Halligan et al., J. Biol. Chem. 260, 2475 (1985).Plasmid YEpG was also purified by the alkali lysis method followed byphenol deproteination and CsCl/ethidium isopycnic centrifugation asdescribed by T. Mariatis et al., Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Labs, N.Y. (1982) at pages 149-185. The end-labelingof the plasmid was accomplished as previously described by L. F. Liu etal., J. Biol. Chem., 258, 15365 (1983). The cleavage assays wereperformed as previously reported by A. Y. Chen et al., Cancer Res. 53,1332 (1993). Human topoisomerase was isolated as a recombinant fusionprotein using a T7 expression system.

B. Cytotoxicity assay

The cytotoxicity was determined using the as MTT-microtiter platetetrazolinium cytotoxicity assay (MTA) following the procedures of F.Denizot et al., J. Immunol. Methods, 89, 271 (1986); J. Carmichael etal., Cancer Res., 47, 936 (1987) and T. J. Mosmann et al., Immunol.Methods, 65, 55 (1983). The human lymphoblast RPMI 8402 and itscamotothecin-resistant variant cell line, CPT-K5 were provided by Dr.Toshiwo Andoh (Aichi Cancer Center Research Institute, Nagoya, Japan).See, for example, T. Andoh et al., Adv. Pharmacol., 29B, 93 (1994). Thecytotoxicity assay was performed using 96-well microtiter plates. Cellswere grown in suspension at 37° C. in 5% CO₂ and maintained by regularpassage in RPMI medium supplemented with 10% heat inactivated fetalbovine serum, L-glutamine (2 mM), penicillin (100 U/ml), andstreptomycin (0.1 mg/ml). For determination of IC₅₀, cells were exposedcontinuously with varying concentrations of drug concentrations and MTTassays were performed at the end of the fourth day.

The drug sensitive human epidermoid carcinoma KB3-1 cell line (S.Aliyama et al., Somatic Cell Mol. Genet., 11, 117 (1985)) and itsvinblastine-selected multidrug-resistant variant KBV-1 cells (D. W. Shenet al., Science, 32, 643 (1986)) were provided by Dr. Michael Gottesmann(National Cancer Institute, Bethesda, Md.). These cells were grown asmonolayer cultures at in 5% CO₂ and maintained by regular passage inDulbecco's minimal essential medium supplemented with 10% heatinactivated fetal bovine serum. KBV-1 cells were similarly maintainedexcept they were grown in the presence of 1 μg/ml vinblastine.

C. Results

As shown on Table 1, comparison of compounds 10-16 with Hoechst 33342(1) as inhibitors of topoisomerase I demonstrated that several of thesetrisbenzimidazoles had similar potency.

                  TABLE 1                                                         ______________________________________                                        Topoisomerase I-mediated DNA Cleavage and Cytotoxicity                        of Bis- and Trisbenzimidazoles                                                       Topo I-  Cytotoxicity IC.sub.50.sup.a (μM)                                 mediated Cell Lines                                                    Compound DNA cleavage.sup.b                                                                       RPMI    CPT-K5 KB3-1 KBV-1                                ______________________________________                                        Hoechst 33342                                                                          1          0.03    0.9    0.01  1.2                                  10       1.1        14      28     N.D.  N.D.                                 11       1          >25.sup.c                                                                             >25.sup.c                                                                            N.D.  N.D.                                 12       100        7.6     20     N.D.  N.D.                                 13       2          0.09    0.58   0.58  0.35                                 14       3.3        0.16    5.8    0.05  0.09                                 15       2          0.035   2.5    0.02  0.02                                 16       2          0.035   2.5    0.02  0.01                                 19       1000       >25.sup.c                                                                             N.D.   N.D.  N.D.                                 ______________________________________                                         .sup.a IC.sub.50 has been calculated after 4 days of continuous drug          exposure. N.D. = Not determined.                                              .sup.b Topoisomerase I cleavage values are reported as REC, Relative          Effective Concentration, i.e. concentrations relative to Hoechst 33342,       whose value is arbitrarily assumed as 1, that are able to produce the sam     cleavage on the plasmid DNA in the presence of calf thymus topoisomerase      I. Cleavage is calculated from the intensity of the strongest Hoechst         specific band.                                                                .sup.c No indication of cytotoxicity were considered indicative of            IC.sub.50 values substantially greater than the highest doses assayed.   

While 10 and 11 exhibited similar potency in their inhibition oftopoisomerase I as observed with Hoechst 33342, both of these compoundsfailed to exhibit significant cytotoxicity towards the human lymphoblastcell line, RPMI 8402. However, this may be due to the inability of thepure compound to penetrate the target cells, which may be overcome byselection of a suitable carrier, such as liposomes. The 5-phenylsubstituted trisbenzimidazole, 13, was approximately one-half as potentas Hoechst 33342 as a topoisomerase I inhibitor. In contrast to 10 and11, however, it had significant cytotoxicity towards the humanlymphoblast cell line, RPMI 8402 cells. As observed with Hoechst 33342,13 was also effective against camptothecin-resistant CPT-K5 cells. Therelative resistance of Hoechst 33342 and 13, expressed as the ratio ofthe IC₅₀ values of the resistant verses the drug sensitive cell line, isapproximately 30 fold as compared to the relative resistance ofcamptothecin which is 2,500 fold, as reported by A. Y. Chen et al.,Cancer Res., 53, 1332 (1993). A similar effect was observed in anotherpair of cell lines; 13 has an IC₅₀ of 0.015 μg/ml in the human ovariantumor cell line, A2780, relative to an IC₅₀ of 0.03 μg/ml in CPT-2000, avariant of A2780 selected for camptothecin-resistance and known tocontain a mutant camptothecin-resistant topoisomerase I. The 5-n-propyltrisbenzimidazole derivative, 12, was much less active than either 10,11, or 13 as an inhibitor of topoisomerase I. Its weak activity as atopoisomerase I inhibitor correlated with its weak cytotoxicity. Theactivity of several of these compounds were also evaluated usingrecombinant human topoisomerase I. Several of these analogs inducedsimilar DNA cleavage in the presence of human topoisomerase I ascompared to that observed with topoisomerase I isolated from calfthymus.

The cytotoxic activity of Hoechst 33342 and 13 was also evaluatedagainst KB 3-1 and KB V-1 cells. The primary difference between thesecell lines is in the degree to which human MDR1 (P-glycoprotein) isexpressed. Recent studies have demonstrated that antineoplastic agentswhich are cationic at physiological pH are more likely to serve assubstrates for MDR1 and, therefore, are likely to be less effectiveagainst cells that overexpress P-glycoprotein. In view of the fact thatHoechst 33342 is extensively protonated at physiological pH, it is notsurprising that the IC₅₀ differs by approximately two-orders ofmagnitude for KB 3-1 as compared to KB V-1 cells, as reported by A. Y.Chen et al., Adv. Pharmacol., 245, 29B (1994). In contrast to Hoechst33342, there is little difference between the IC₅₀ values observed for13 in these two cell lines. Thus, 13 appears not to be a substrate forhuman MDR1. This data indicate that these trisbenzimidazole derivativesmay have significant chemotherapeutic advantages as compared to Hoechst33342 or pibenzimol (Hoechst 33258),2'-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimidazole.

These data indicate that substitution of these trisbenzimidazole with a5-Ar substituent can yield derivatives which are active as topoisomeraseI inhibitors and cytotoxic to tumor cells. Trisbenzimidazolessubstituted at the 5-position with either a 2-, 3-, or 4-pyridyl group,14-16, were evaluated for their potency as topoisomerase I inhibitorsand for cytotoxicity as summarized in Table 1. These analogs, similar to13, have activity as topoisomerase I inhibitors. The 3- and 4-pyridylanalogs, 15 and 16, are somewhat more active than the 2-pyridylderivative, 14, as topoisomerase I inhibitors as well as cytotoxicagents. As was observed with 13, these pyridyl-substitutedtrisbenzimidazoles had similar cytotoxicity to KB 3-1 cells as well asto KB V-1 cells which overexpress MDR1. A principal advantage of theseheteroaryl substituted trisbenzimidazoles as compared to Hoechst 33342is their efficacy against cell lines which express MDR1.

All publications and patents are incorporated by reference herein, asthough individually incorporated by reference. The invention has beendescribed with reference to various specific and preferred embodimentsand techniques. However, it should be understood that many variationsand modifications may be made while remaining within the spirit andscope of the invention.

What is claimed is:
 1. A compound of the formula: ##STR5## wherein Ar is(C₆ -C₁₂)aryl or (5- or 6-membered) heteroaryl comprising 1-2N, S, ornonperoxide O, wherein N is unsubstituted or is substituted with (C₁-C₄)alkyl; X is H, CN, CHO, OH, acetyl, CF₃, O(C₁ -C₄)alkyl, NO₂ or NH₂; each of Y is H, (C₁ -C₄)alkyl or aralkyl; Y' is H or (C₁ -C₄) alkyl; nis 1; or a pharmaceutically acceptable salt thereof.
 2. The compound ofclaim 1 wherein Ar is at the 5-position.
 3. The compound of claim 1wherein Ar is phenyl or pyridyl.
 4. The compound of claim 3 whereinpyridyl is 2-pyridyl, 3-pyridyl or 4-pyridyl.
 5. The compound of claims3 or 4 wherein X is H.
 6. The compound of claim 5 wherein Y' is H. 7.The compound of claims 3 or 4 wherein each Y is H.
 8. A compound of theformula: ##STR6## X is CN, CHO, OH, acetyl, CF₃, O(C₁ -C₄)alkyl, NO₂ orNH₂ ; each of Y is H, (C₁ -C₄)alkyl or aralkyl; Y' is H or (C₁-C₄)alkyl, and n is 0; or a pharmaceutically acceptable salt thereof. 9.The compound of claim 8 wherein X is CHO or CN.
 10. The compound ofclaims 8 or 14 wherein Y' is H.
 11. The compound of claims 8, 9 or 10wherein each Y is H.
 12. A therapeutic composition comprising an amountof the compound of claims 1 or 8 in combination with a pharmaceuticallyacceptable carrier effective to inhibit mammalian tumor cell growth.